Placental tissue composition for for treating cardiac tissue damage

ABSTRACT

Described herein are compositions and methods of treating a cardiac condition using modified placental tissue or an extract of a placental tissue, capable of recruiting stem cells or promoting healing in vivo and in vitro.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/158,757, filed Jan. 17, 2014, which claims the benefit of U.S.Provisional Patent Application No. 61/849,838, filed on Jan. 18, 2013,each of which is hereby incorporated by reference in its entirety.

BACKGROUND Field of the Invention

This invention is directed, in part, to methods of treating cardiacconditions by use of a sufficient amount of modified placental tissue oran extract of a placental tissue.

State of the Art

Despite advances in patient care and treatment, cardiovascular diseasesremain the biggest cause of deaths worldwide.

There are about 6 million Americans living with heart failure (HF), andthere are 670,000 new cases of HF each year. HF is the primary reasonfor 12-15 million office visits and 6.5 million hospital days annually.The economic burden and overall healthcare impact of HF are staggering.Indeed, it is estimated that the total direct and indirect costs for HFin the United States exceed $30 billion each year. Remarkably,atherosclerotic coronary artery disease (CAD) accounts for 60-75% of allsymptomatic HF, with the history of MI conferring an increased relativerisk of 6.0 for HF.

Therefore, there is need for additional treatment options forcardiovascular diseases, such as heart failure.

SUMMARY OF THE INVENTION

Cardiac conditions by and large involve damaged cardiac tissue such asdamage cardiac muscles arising from ischemic events, damages to the leftventricle such as those arising from congested heart failure, damages tothe heart valves arising from diseases, such as coronary arterydiseases. In all aspects, improving function of the damaged portion ofthe cardiac tissues would be a benefit to the patient. Conventionaltreatments include introducing a mechanical device, such as a stent, anexternal pump, and the like, to treat the disease or the condition.Other treatment conditions include coronary bypass procedures where thecoronary arterial blockage is circumvented by introduction of anautologous vessel such as a leg vein which is connected to the artery byanastomosis.

This invention is directed to the discovery that modified placentatissue, possesses numerous biological factors that induce the patient'shealing of the damaged organ or tissue. Such factors include stem cellrecruiting factors, growth factors and angiogenesis inducing factors,all of which either alone or in combination, interact to minimize damageand to heal already damaged cardiac tissue.

Accordingly, in one aspect, there is provided a method for treating acardiac condition in a patient in need thereof, comprising administeringto said patient a sufficient amount of a composition comprising amodified placental tissue or an extract of a placental tissue.

In one aspect, provided is a composition comprising modified placentaltissue configured for non-obstructive placement to an area approximateto a damaged cardiac tissue in an amount sufficient to minimize damageand induce healing. For example, the placenta tissue may be introducedat or approximate to the damage, to induce healing of the tissue, bybiological processes including angiogenesis. In some embodiments, themodified placental tissue retains an effective amount of stem cellrecruiting factors, growth factors, and/or angiogenesis inducingfactors.

In another aspect, provided is a method for treating damaged or diseasedcardiac tissue, which method comprises placing an effective amount of amodified placental tissue or an extract of a placental tissueapproximate to the damaged cardiac tissue without obstructing thefunction thereof, wherein the modified placental tissue or the extractis placed under conditions that promote treatment of the disease orhealing of the damaged tissue.

In another aspect, provided is a method for treating a cardiac conditionin a patient in need thereof comprising providing to an area proximateto the heart of the patient with a composition comprising a modifiedplacental tissue or an extract of a placental tissue comprising aneffective amount of stem cell recruiting factors, wherein said modifiedplacental tissue or extract, when placed in contact with said area,promotes stem cell recruitment to said area. In one embodiment, the stemcell recruited is a hematopoietic stem cell (HSC). In anotherembodiment, the stem cell recruited is a mesenchymal stem cell (MSC). Inone embodiment, the stem cell recruited is bone marrow-derived stemcells.

In a related aspect, the compositions encompassed by the presentinvention are used for inducing angiogenesis to treat conditions otherthan cardiovascular conditions. This aspect is described in U.S.Provisional Patent Application No. 61/928,986, filed on even date under,titled “Method for Inducing Angiogenesis,” the content of which isincorporated by reference in its entirety.

These and other aspects of this invention are further described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee. The accompanying drawings, which are incorporatedin and constitute a part of this specification, illustrate severalaspects described below.

FIG. 1 shows a schematic for a cell culture insert for stem cellmigration assays described in Example 3.

FIG. 2 shows a bar graph of percent cell migration in human mesenchymalstem cells (MSCs) cultured in the presence of various amounts ofEpiFix®. Details are described in Example 3.

FIG. 3A shows a bar graph of percentage living/Lin⁻ mouse hematopoieticstem cells in normal skin, sham implant, acellular dermal matrix, andEpiFix® at 3, 7, 14, and 28 days post implant. Values shown aremeans+/−standard deviation, n=4 specimens. ** indicates p<0.05 whencomparing EpiFix® or control ADM to normal skin and sham implant viaone-way ANOVA. †† indicates p<0.05 when comparing EpiFix® to control ADMvia two tailed t-test. FIG. 3B shows a bar graph of percentageliving/Lin⁻ mouse mesenchymal cells in normal skin, sham implant,acellular dermal matrix, and EpiFix® at 3, 7, 14, and 28 days postimplant. Values shown are means+/−standard deviations, n=4 specimens. **indicates p<0.05 when comparing EpiFix® or control ADM to normal skinand sham implant via one-way ANOVA. Details are described in Example 4.

FIGS. 4A and 4B demonstrate that AmnioFix® and EpiFix® membraneprotected mice from damage following experimentally induced myocardialinfarction (MI). Treatment of mice with either the AmnioFix® or EpiFix®membranes significantly reduced infarct size on NOD/SCID and wild-typemice following MI. Similar levels of protection were seen for eachmembrane type. FIG. 4A shows quantitation of infarct size relative tototal area of the heart from membrane treated mice and control treatedmice is shown. Three sections were taken of each heart and quantitationof infarct size was performed on the two sections proximal to the siteof infarct. The sections were stained with Masson's trichrome. FIG. 4Bshows representative images of membrane treated and control treatedwild-type and NOD/SCID mice. AmnioFix® membrane=membrane 2, 3, 4 and 6;EpiFix® membrane=membrane 1, 5 and 7.* p<0.05, ** p<0.001. Details aredescribed in Example 6.

FIGS. 5A-5C demonstrate that treatment with the AmnioFix® and EpiFix®membrane increased the number of c-kit+ cells. FIG. 5A shows schematicdepiction of the placement of the AmnioFix® or EpiFix® membrane. FIG. 5Bshows representative immunohistochemical staining for c-kit for regionsof the heart covered by the AmnioFix® or EpiFix® membrane or regionsperipheral to the membrane. FIG. 5C shows quantitation of c-kit+ cellsin the tissue sections. Details are described in Example 6.

FIGS. 6A and 6B demonstrate that Ki67 levels were elevated inmembrane-treated heart tissue both in the area covered by the membraneand outside of the membrane-treated area. FIG. 6A shows representativeimmunocy to chemical staining of membrane and control-treated hearts.The treated hearts were stained for Ki-67 (green) and the cardiac musclecell marker troponin, Troponin C (red). DAPI is in blue. FIG. 6B showsquantitation of the Ki-67+ cells from the membrane treated compared tocontrol treated hearts. Details are described in Example 6.

FIGS. 7A and 7B demonstrate that the membrane-treated hearts hadsignificantly increased numbers of blood vessels compared tocontrol-treated tissue. FIG. 7A shows CD31 staining (green) of themembrane treated and control treated hearts indicated a significantincrease in the number of blood vessels. The sections were co-stainedfor troponin (red) and DAPI (Blue). FIG. 7B shows quantitation of meannumber of vessels in membrane (EpiFix® and AmnioFix®) treated heartscompared to saline treatment (control). Details are described in Example6.

FIGS. 8A and 8B demonstrate that membrane treatment significantlydecreased apoptosis following MI. FIG. 8A shows TUNEL staining was usedto identify apoptotic cells in the membrane and control-treated hearttissue. FIG. 8B shows quantitation of TUNEL positive cells in themembrane and control treated cells. Details are described in Example 6.

DETAILED DESCRIPTION Definitions

Before this invention is disclosed and described, it is to be understoodthat the aspects described below are not limited to specificcompositions, synthetic methods, or uses as such may, of course, vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular aspects only and is not intended to belimiting.

In this specification and in the claims that follow, reference will bemade to a number of terms that shall be defined to have the followingmeanings:

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a bioactive agent” includes mixtures of two or more suchagents, and the like.

“Optional” or “optionally” means that the subsequently described eventor circumstance can or cannot occur, and that the description includesinstances where the event or circumstance occurs and instances where itdoes not. For example, the phrase “optionally cleaning step” means thatthe cleaning step may or may not be performed.

The term “subject” or “patient” as used herein refers to any vertebrateorganism including, but not limited to, mammalian subjects such ashumans, farm animals, domesticated pets and the like.

The term “amnion” as used herein includes amniotic membrane where theintermediate tissue layer is intact or has been substantially removed.

The term “exterior surface” refers to either or both surfaces of themodified placental tissue which will contact the diseased or injuredcardiac tissue of the patient to which the placental tissue is applied.

The term “cardiac tissue” as used herein refers to any part of the heartincluding the pericardium, the endocardium, the myocardium and theepicardium, as well as blood vessels and nerves on or connected to theheart, etc.

The term “diseased” as used herein refers to an organ and/or body part,such as a cardiac tissue, that is characterized as being in a diseasestate, or susceptible to being in a disease state.

The term “injured” as used herein is used to have an ordinary meaning inthe art, and includes any and all types of damage to an organ and/orbody part, such as a cardiac tissue.

The term “biocompatible” as used herein refers to a material that issuitable for implantation or injection into a subject. In variousaspects, a biocompatible material does not cause toxic or injuriouseffects once implanted in the subject.

The term “modified placental tissue” refers to any and all components ofplacental tissue including whole placental tissue that has been modifiedby cleaning, disinfecting, and/or segmenting the tissue as well as toseparated components of placental tissue such as amnion, chorion, theumbilical cord, and the like. Modified tissue may maintain cellularlayers, such as the epithelial layer and/or the fibroblast layer.Modified placental tissue may include further modification, such aslamination of one or more layers of placental tissue, micronization ofplacental tissue, chemisorption or physisorption of small molecules,proteins (e.g. growth factors, antibodies), nucleic acids (e.g.aptamers), polymers, or other substances.

The term “extract of a placental tissue” refers to a composition, suchas a solution or a lyophilized solid, comprising one or more of thebiological factors present in a placental tissue or modified placentaltissue and substantially free of the placental tissue or cell materials.Such extracts included those described in U.S. patent application Ser.No. 13/744,331, filed on Jan. 17, 2013, converted to U.S. ProvisionalPatent Application No. 61/956,185, titled “NON-SURGICAL, LOCALIZEDDELIVERY OF COMPOSITIONS FOR PLACENTAL GROWTH FACTORS,” and U.S. patentapplication Ser. No. 13/745,642, filed on Jan. 18, 2013, titled“ISOLATED PLACENTAL STEM CELL RECRUITING FACTORS”, and can be preparedaccording to methods described therein. Both of patent applications arehereby incorporated by reference in their entirety.

The term “sufficient amount” or “effective amount” refers to an amountof a 15 modified placental tissue or an extract of a placental tissuethat is sufficient to inhibit, reduce or minimize a cardiac damage orinduce healing of the diseased or injured cardiac tissue over time,either in vivo or in vitro. The “sufficient amount” will vary dependingon a variety of factors, such as but not limited to, the type and/oramount of the placental tissue or extract of a placental tissue used,the type and/or size of the diseased or injured cardiac tissue to betreated, the severity of the disease or injury to the diseased orinjured cardiac tissue to be treated and the administration route. Thedetermination of a “sufficient amount” can be made by one of ordinaryskill in the art based on the disclosure provided herein.

The term “placental growth factors” refers to that array of growthfactors obtainable from modified placental tissue. The manner ofobtaining such growth factors is not critical to the invention andinclude, by way of example only, aqueous extraction from the placenta,culturing of placental cells expressing such growth factors, and thelike. The concentration of extracted growth factors can be increased byreducing the volume of water, saline, or buffer used to extract thegrowth factors, by addition of growth factors produced from placentalcell cultures, and the like.

The term “stem cell recruiting factors” refers to any and all factorsthat are capable of inducing the recruitment of stem cells and causingthem to migrate towards a source of such factors. Non-limiting examplesof stem cell recruiting factors may be one or more CC chemokines, CXCchemokines, C chemokines, or CX₃C chemokines.

The term “stem cell recruitment” refers to direct or indirect chemotaxisof stem cells to a modified placental tissue or an extract of aplacental tissue. In one aspect, the recruitment may be direct, whereinstem cell recruiting factors (e.g. chemokines, which induce cellchemotaxis) in a modified placental tissue are released from theplacental tissue or extract comprising such factors are introduced tothe site to be treated and which induce stem cells to migrate towardsthe site. In another aspect, the recruitment may be indirect, whereinstem cell recruiting factors alone or in a modified placental tissueinduce nearby cells to release factors (e.g. chemokines), that in turninduce stem cells to migrate towards the placental tissue. Stillfurther, stem cell recruitment may embody both direct and indirectfactors.

The term “proximate to” as used herein means adjacent to, or contactinga body part, such as a diseased or injured cardiac tissue, such that thecomposition exerts the desired effect. In general, “proximate to” meansa distance that is generally within the skill of the art but preferablyis within 3 cm, 2 cm, or 1 cm of the organ or body part, including on orin the body part. The term “contact” or “contacting” means that thecomposition is on or in the body part.

The term “exogenous” refers to substances that are not naturallyoccurring to a body part being treated, including allograft tissue, suchas modified placental tissue.

The term “endogenous” refers to autologous biological substances from asubject.

As used herein, the term “bioerodible,” which is used hereininterchangeably with the term “biodegradable,” refers to a biocompatiblematerial that gradually decomposes, dissolves, hydrolyzes and/or erodesin situ, or that is susceptible to degradation into smaller componentsor molecules in a living organism over a prolonged period of time, forexample, over days or months, such that the material is harmless to theliving organism under normal living conditions. Generally, the“bioerodible” polymers herein are polymers that are hydrolyzable, andbioerode in situ primarily through hydrolysis. Preferably, the smallercomponents or molecules are biocompatible to a patient.

As one of ordinary skill in the art would understand, the degradation ofthe material results in a continuous release of a therapeutic amount ofplacental growth factors incorporated in the material over a prolongedperiod of time, such as about 3 days, about 5 days, about 10 days, about15 days, about 20 days, about 25 days, about 30 days, about 2 months,about 3 months, about 4 months, about 5 months, or about 6 months. Adesired release rate can be determined and/or achieved by adjusting theinitial concentration of the growth factors incorporated in thebioerodible or biodegradable mass and the degradation rate of the mass.

Titles or subtitles may be used in the specification for the convenienceof a reader, which are not intended to influence the scope of thepresent invention. Additionally, some terms used in this specificationare more specifically defined below.

Compositions and Methods

When a heart tissue, such as heart muscle, is damaged, for example, in aheart attack, stem cells can be recruited to make repairs. However, itis believed that the heart has a tendency to stop signaling for stemcells before the repair is complete. As a result, the damaged tissue maybe only partially repaired, which becomes a burden to the heart, forcingit to work harder and less efficiently, and possibly leading toadditional harmful effect to the heart. Further, current therapeuticinjections of stem cells into the damaged tissue are complicated by thelow rate of survival of injected stem cells.

This invention is based, in part, on the discovery that application of asufficient amount of a composition comprising a modified placentaltissue, such as amnion, or an extract of a placental tissue, proximateto a diseased or injured cardiac tissue of a patient not only providesan exogenous treatment regimen but surprisingly also provides anendogenous therapeutic effect through eliciting, directly or indirectly,stem cell recruitment to the site of the diseased or injured body part,thereby achieving a binary therapeutic effect. In the case of placentaltissue, it is contemplated that administration of the modified placentaltissue to the diseased or injured cardiac tissue not only provides aphysical remedy and/or barrier to prevent undesired tissue adhesion butalso induces an endogenous effect by improving the efficiency of stemcell recruitment and/or recruits stem cell when the heart itself failsto do so. It is further contemplated that the modified placental tissueor extract stimulates growth and healing of the diseased or injuredtissue by providing growth factors and/or angiogenesis inducing factorsto the diseased or injured tissue, separately or in combination withinducing stem cell recruitment.

Accordingly, in one aspect, provided is a composition comprising aplacental tissue or factors extracted from a placental tissue which isdelivered for the non-obstructive 10 placement to an area approximate toa diseased or injured cardiac tissue in an amount sufficient to reducedamage and induce healing. In some embodiments, the modified placentaltissue or an extract of a placental tissue comprises an effective amountof stem cell recruiting factors, growth factors, and/or angiogenesisinducing factors. In some embodiments, the placenta tissue or an extractof a placental tissue induces healing of the tissue by biologicalprocesses including angiogenesis.

In another aspect, there is provided a method for treating a cardiaccondition in a patient in need thereof, comprising administering to saidpatient an sufficient amount of a composition comprising a modifiedplacental tissue or an extract of a placental tissue. In someembodiments, the composition is administered by being placed approximateto a cardiac tissue having or causing the cardiac condition.

In another aspect, provided is a method for treating diseased or injuredcardiac tissue, which method comprises placing an effective amount of amodified placental tissue or an extract of a placental tissue configuredto being placed approximate to the diseased or injured heart tissuewithout obstructing the function thereof, wherein the modified placentaltissue or extract of a placental tissue is placed under conditions thatpromote treatment of the disease or healing of the injured tissue.

In another aspect, provided is a method for treating a cardiac conditionin a patient in need thereof comprising providing to an area proximateto the heart of the patient with a composition comprising a modifiedplacental tissue or an extract of a placental tissue which comprises aneffective amount of stem cell recruiting factors, growth factors andangiogenesis inducing factors, wherein said modified placental tissue orextract, when placed in contact with or proximate to said area, promotesstem cell recruitment to said area and/or healing of the cardiac tissue.In one embodiment, the stem cell recruited is a haematopoietic stem cell(HSC). In another embodiment, the stem cell recruited is a mesenchymalstem cell (MSC). In another embodiment, the stem cell recruited is bonemarrow derived stem cell.

In another aspect, provided is a method for treating a cardiac conditioncomprising a diseased or injured cardiac tissue in a patient in needthereof, comprising contacting said cardiac tissue with a sufficientamount of a composition comprising a modified placental tissue or anextract of a placental tissue which comprises an effective amount ofstem cells recruiting factors, growth factors and angiogenesis inducingfactors.

In some embodiments, the cardiac condition is selected from aninflammatory heart condition, such as myocarditis, pericarditis andendocarditis, and a necrotizing condition.

In some embodiments, the cardiac condition is selected from acutemyocardial infarction, myocardial infarction, cardiomyopathy (e.g.,ischemic cardiomyopathy, myocarditis or inflammatory cardiomyopathy,dilated cardiomyopathy, and hypertrophic cardiomyopathy), unstableangina, refractory angina, heart attack, heart failure, corpulmonale,vein graft diseases such as degeneration and occlusion of saphenous veingrafts, coronary heart diseases, occlusive coronary thrombus (e.g.,thrombus occurring post-thrombolytic therapy or post-coronaryangioplasty). In some embodiments, the cardiac condition is inflammationof a heart valve. In some embodiments, the cardiac condition is selectedfrom valvular heart disease, inflammatory cardiomegaly, andatherosclerosis.

Angina is severe chest pain due to ischemia (a lack of blood, thus alack of oxygen supply) of the heart muscle, generally due to obstructionor spasm of the coronary arteries (the heart's blood vessels). Coronaryartery disease, the main cause of angina, is due to atherosclerosis ofthe cardiac arteries. Various open cardiac and vascular surgeryprocedures to remove atherosclerotic clots require the repair,reconstruction and closure of the vessel, and the support of aregenerative tissue patch to close and patch the surgical defect. Heartby-pass grafts and heart defect reconstruction (as part of an open-heartsurgical procedure) also can benefit from a patch or graft to provide abuttress to soft-tissue weakness, tissue replacement if there is a lackof suitable tissue, and also the potential to reduce adhesions to theheart itself. The modified placental tissue can be used as a patch tosupport the repair of vascular and cardiac defects caused by operationsand complications such as carotid artery repair, coronary artery bypassgrafting, congenital heart disease, heart valve repair, and vascularrepair (i.e. peripheral vessels).

In some embodiments, the method is for promoting healing of a surgicalsite of the heart and/or reducing scarring. In some embodiments, themethod is for promoting tissue regrowth after a cardiac surgery, such asa valve repair or anastomosis.

In some embodiments, the method is for treating a pericardial disease,such as acute pericarditis. In some embodiments, the method is usedduring and/or after replacement of pericardium. In some embodiments, themethod is for treating Dresslers syndrome or reducing inflammation inDresslers syndrome.

In some embodiments, the placental tissue attracts endogenous stem cellsand provide a microenvironment for stem cell survival.

In some embodiments, the placental tissue is preconditioned in vitro, soas to facilitate the proliferation and optional differentiation of theexogenous stem cells into cardiomyogenic lineage.

In some embodiments, the placental tissue is a biocompatible andnontoxic support material for the stem cells.

In some embodiments, the placental tissue material is a patch, andrepresents a structurally resistant element that serves as a bandage forthe diseased or injured area. More specifically, the placental tissuematerial functions as a molecular and mechanical bandage, serving aschemoattractant for endogenous stem cells (e.g., cardiac and/or bonemarrow) to migrate to the site of injury and stimulating theirproliferation to promote healing, therefore, reduce scar tissueformation and inhibiting inflammation.

In some embodiments, the placental tissue material is integrated withinthe host tissue and optionally replaced by the host extracellular matrix(ECM).

In one embodiment, placental tissue may be modified as described in U.S.Ser. No. 61/683,698, including cleaning, separation of the amnion andchorion, removal or maintenance of the epithelial cell layer,decontamination, and dehydration. Dehydration may be accomplished usingthe drying apparatus or chemical dehydration, for example, as describedin U.S. Ser. No. 13/691,509, filed Nov. 30, 2012. Both of whichapplications are incorporated herein by reference in their entirety.Each aspect of that process produces modified placental tissue for thepurposes of this invention whether used alone or in combination.However, it is preferred that the modified placental tissue include atleast the steps of cleaning and decontamination. As such, modifiedplacental tissue preferably comprises placental tissue which has beencleaned and decontaminated and also includes placental tissue which hasundergone one or more of separation of the amnion and chorion, removalof the epithelial cell layer, and dehydration. Modified placental tissuecan also be formed into layers which may be dried separately andlaminated together or dried together to form multi-layer laminates.

Modified placental tissue may also be micronized into particles of avariety of sizes, for example, no more than about 300 microns in size,such as less than about 250 microns, less than about 200 microns, lessthan about 150 microns, less than about 100 microns, or less than about50 microns. Micronized placental tissue may be sandwiched between one ormore layers of a multilayer laminate, or on top of a laminate.Micronized placental tissue may also be added to single layer ofmodified placental tissue. See, for example, International PatentApplication No. PCT/US2012/024798, filed Feb. 13, 2012, as well as U.S.Provisional Patent Application Ser. No. 61/683,700 filed Aug. 15, 2012both of which are incorporated herein by reference in their entirety. Itis also contemplated that micronized modified placental tissue canenhance the rate of stem cell recruitment in a particular body part. Insome embodiments, micronized modified placental tissue is added tomodified placental tissue, either a single layer of modified placentaltissue, or in between a multi-layer laminate of placental tissue.

In some embodiments, the modified placental tissue is selected fromamnion, chorion, or both amnion and chorion. In some embodiments,modified placental tissue does not include the umbilical cord.

Amnion is a unique ECM due to the presence of collagen types IV, V andVII, which enables the amnion to bind water and swell. The intermediatetissue layer of the amniotic membrane is composed largely ofglycoproteins and proteoglycans, which also enables the intermediatetissue layer to bind water. Thus, the modified placental tissue whenapplied to a diseased or injured cardiac tissue helps retain water atthat site, which facilitates cardiac tissue repair and/or regeneration.For example, cell migration, including stem cell recruitment, within thehealing cascade is facilitated in a hydrophilic environment. Theintermediate layer is also composed of collagen types I, III, and IV.Type I collagen provides mechanical strength to skin by providing amajor biomechanical scaffold for cell attachment and anchorage ofmacromolecules. Type III collagen provides elasticity.

In other embodiments, the epithelial cell layer of the amnion is notremoved. In these embodiments, the modified placental tissue providesadditional protection by maintaining separation from the peritoneum,larger vessels, and abdominal musculature. The modified placental tissuemay serve as a reduced friction anatomical barrier against adhesions andscaring. In one embodiment, the application of the modified placentaltissue described herein where the epithelial layer of the skin isdisrupted can be effective in delivering the growth factors directly tothe injured site to promote healing and/or stem cell recruitment.

In some embodiments, the epithelium of the amnion is substantiallyremoved. Removal of the epithelial cell layer exposes the amnion'sbasement membrane layer, which increases cell signaling characteristics.This up regulation response enhances cellular migration and expressionof anti-inflammatory proteins, which inhibits fibrosis.

In some embodiments, the composition comprising a modified placentaltissue or extract of a placental tissue is provided to an area adjacentto a diseased or injured cardiac tissue via injection of a compositioncomprising a micronized modified placental tissue or the extract, viasurgical implantation of the composition or via patch delivery of thecomposition.

In some embodiments, the composition comprising a modified placentaltissue or extract is in an injectable form comprising micronizedmodified placental tissue or extract and a pharmaceutically acceptablecarrier. Suitable pharmaceutically acceptable carriers include, but arenot limited to, such as water, a buffer (e.g., phosphate buffered saline(PBS), citrate buffer, etc.), water-soluble organic solvents (e.g.,polyethylene glycol 300, polyethylene glycol 400, ethanol, propyleneglycol, glycerin, N-methyl-2-pyrrolidone, dimethylacetamide, anddimethylsulfoxide), organic liquids/semi-solids (beeswax, d-tocopherol,oleic acid, medium-chain mono- and diglycerides), non-ionic surfactants(polyethoxylated castor oils (e.g., Cremophor EL, Cremophor RH 40,Cremophor RH 60), polysorbate 20, polysorbate 80, poloxamer 188,poloxamer 407, d-tocopherol polyethylene glycol 1000 succinate,polyethylene glycol (15)-hydroxystearate, sorbitan monooleate, oleoylpolyoxyl-6 glycerides, linoleoyl polyoxyl-6 glycerides, caprylocaproylpolyoxyl-8 glycerides, Gellucire® 44/14, Softigen® 767, and mono- anddi-fatty acid esters of PEG 300, 400, or 1750, etc.), a lipid (e.g.,castor oil, corn oil, cottonseed oil, olive oil, peanut oil, peppermintoil, safflower oil, sesame oil, soybean oil, hydrogenated vegetableoils, hydrogenated soybean oil, and medium-chain triglycerides ofcoconut oil and palm seed oil), cyclodextrin (such as α-cyclodextrin,β-cyclodextrin, and γ-cyclodextrin, hydroxypropyl-β-cyclodextrin, andsulfobutylether-(β-cyclodextrin), and phospholipids (e.g.,phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,distearoylphosphatidylglycerol, 1-dimyristoylphosphatidylcholine,1-dimyristoylphosphatidylglycerol, etc.), or a mixture thereof.

In some embodiments, the composition forms a localized mass when appliedto or proximate to said diseased and/or injured cardiac tissue with anagent which allows for localized retention of the solution or suspensionat the site of delivery optionally for extended and/or continuousrelease of the biological factors in the composition. Such agentsinclude 25 thixotropic agents, phase changing agents, and the like.These compositions are in an injectable form at ambient conditions andform a viscous or gel-like bioerodible or biodegradable mass in vivowhich limits transport away from the site of delivery and allows for thediffusion of the biological factors from the mass formed over a periodof time.

In some embodiments, localization agents, such as thixotropic agents,phase changing agents, and the like, may include but not limited to,hydrogel, bioerodible, biocompatible polymer, and collagen gels. Thepresence of one or more localization agents in the compositions of thisinvention allows the compositions to have certain viscosity such thatthe compositions are locally retained for a period of time uponadministration or injection. It is within the purview of one of ordinaryskill in the art to determine the suitable viscosity of thecompositions. In some aspects, the compositions have a viscosity betweenabout 5 cP to about 1×10⁸ cP, or about 5 cP to about 1×10⁶ cP, or about5 cP to about 1×10⁵ cP, or about 5 cP to about 1×10⁴ cP, or about 5 cPto about 1×10³ cP, or about 6 cP to about 9500 cP at 10 25° C.

The hydrogels useful in the compositions of this invention can bechemically and/or physically cross-linked hydrogels. In situ chemicalcross-linking is obtained, e.g., via photo-initiated, redox-initiated orMichael-type addition polymerization that preferably involve covalentbond formation. Physically cross-linked hydrogels self-assemble underexternal stimuli and do not rely on covalent bond formation.Temperature, pH, ion concentration, and hydrophobic interactions arecertain of the external stimuli useful for such self-assembly and forthe immobilization of such hydrogels.

Exemplary polymers suitable for the use in the composition of thepresent invention include polylactides, polyglycolides,poly(caprolactone), polyanhydrides, polyamines, polyesteramides,polyorthoesters, polydioxanones, polyacetals, polyketals,polycarbonates, polyphosphoesters, polyorthocarbonates,polyphosphazenes, succinates, poly(malic acid), poly(amino acids),polyvinylpyrrolidone, polyethylene glycol, polyhydroxycellulose,polyphosphoesters, polysaccharides, chitin, chitosan, hyaluronic acid,and copolymers, terpolymers and mixtures thereof.

Collagens can be used include, for example, alkaline treatment ofinsoluble collagen extracted from various animals, or by treating withenzyme such as pepsin, trypsin, chymotrypsin, papin or pronase. Thereare no particular restrictions on the origin of the collagen, andtypically collagen can be used that is obtained from the skin, bone,cartilage, tendon or organs, etc. of birds or mammals, Since collagenallows the obtaining of a suitable consistency without heating,preparation can be made easily in the case of gelation. In addition,collagen has a high molecular weight, it more closely resembles livingbody tissue, has considerable physiological activity, and thereforepromotes healing in the case of using on a wound, resulting in a furthertherapeutic effect in combination with the modified placental tissue.Collagen can be flexible after curing and requires only a short time forcrosslinking, in other words, requires only a short time for gelation.Collagen solution can also be made by dissolving in a non-toxic solventrespect to the living body, examples of which include water,physiological saline, a buffer such as borate buffer, or an aqueoussolution containing a salt such as sodium chloride, sodium bromide andpotassium bromide, or protein, sugar or lipid, etc.

The collagen can also form a gel even in the presence of moisture suchas that in blood or humor, and can demonstrate a high degree ofadhesiveness with respect to living body tissue. Collagen solutions usedin the present invention can be made at various concentrations,neutralized and prepared for injection. In various aspects, collagen at0.215 mg/mL, 0.5 mg/mL, 0.75 mg/mL, 1 mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL,5 mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL, 10 mg/mL, 20 mg/mL, 30 mg/mL, 40mg/mL and 50 mg/mL in solution can be used for injection. Upon injectioninto an organ, chilled collagen gels can thermogel as they reach bodytemperature or about 37° C.

Compositions which can form a localized mass with prolonged retention,as well as their preparation including the agents forming thecompositions, such as the phase-changing agents and thixotropic agents,are further described in U.S. patent application Ser. No. 13/744,331,filed on Jan. 17, 2013, converted to U.S. Provisional Patent ApplicationNo. 61/956,185, titled “NON-SURGICAL, LOCALIZED DELIVERY OF COMPOSITIONSFOR PLACENTAL GROWTH FACTORS,” which is hereby incorporated by referencein its entirety.

In some embodiments, the injectable composition is delivered to thecardiac tissue to be treated with needle injection or with a catheter,and the delivery is optionally monitored by ways such as endoscope orfluoroscopy.

In addition to the selection of the components used to make thecomposition comprising a modified placental tissue, the size of themicronized particles present in the grafts can also vary depending upontheir application. In certain aspects, micronized particles having alarger particle size can be used in several applications. For example,the micronized particles (e.g., micronized amnion/chorion tissue graft)having a particle size from 150 um to 350 um can be effective in woundhealing where it is desirable to reduce or prevent scar formation andenhance soft tissue healing.

In some embodiments, the composition comprises modified placental tissuecomprising structural collagens and ECM proteins, regenerative moleculesand/or growth factors. In some embodiments, the composition comprises asufficient amount of growth factors. In some embodiments, the placentalgrowth factors are extracted from modified placental tissue insufficient quantities so as to provide for an aqueous compositioncomprising growth factors optionally without the need to form asuspension with modified placental tissue particles. In anotherembodiment, the composition is free of stem cells or stem cellrecruitment factors. Such compositions can also be formulated so as toform localized mass with prolonged retention in vivo.

Compositions comprising growth factors are further described in U.S.patent application Ser. No. 13/744,331, filed on Jan. 17, 2013,converted to U.S. Provisional Patent Application No. 61/956,185, titled“NON-SURGICAL, LOCALIZED DELIVERY OF COMPOSITIONS FOR PLACENTAL GROWTHFACTORS,” which is hereby incorporated by reference in its entirety.

In some embodiments, the composition comprises modified placental tissuecomprising one or more of platelet derived growth factor AA (PDGF-AA),platelet derived growth factor BB (PDGF-BB), granulocytecolony-stimulating factor (GCSF), TGF_(a), TGFβ, bFGF, EGF, vascularendothelial growth factor (VEGF), keratinocyte growth factor (KGF),hepatocyte growth factor (HGF), nerve growth factor (NGF), heparinbinding epidermal growth factor (HB-EGF), angiogenin, angiopoietin-2(ANG-2), leptin, IL-10, IL-4, placental growth factor (P1GF), TIMP-1,TIMP-2, and TIMP-4. An example of the composition, EpiFix®, has beenshown to stimulate cell migration/proliferation (through a multitude ofgrowth factors), reduce scar tissue (via TIMP5), and inhibitinflammation (through IL-10 and IL-4). It is contemplated that thecomposition can serve as a bandage for the infarct zone. Morespecifically, the placental tissue material functions as a molecular andmechanical bandage, serving as chemoattractant for endogenous stem cells(e.g., cardiac and/or bone marrow) to migrate to the site of injury andstimulating their proliferation to promote healing, therefore, reducescar tissue formation and inhibiting inflammation. The composition isbiodegradable and therefore is non-inflammatory and nontoxic.

In some embodiments, the composition recruits endogenous stem cells andpromote MI healing when used alone. In some embodiments, the compositionenhances cell survival and improve the efficacy of bone marrow stemcells when used together with these cells. In some embodiments, thecomposition is useful in the treatment of MI.

In another aspect, the composition comprising a modified placentaltissue described herein are implanted proximal or internal to a diseasedand/or injured cardiac tissue in an amount sufficient to attract stemcells and promote endogenous healing. In various aspects, in order toattract stem cells to a damaged cardiac tissue, a sufficient amount ofplacental tissue is required before the stem cells migrate to the targetcardiac tissue. For example, as described in Example 3, stem cellsmigration occurred in response to EpiFix® in a concentration-dependantmanner. A 1.5 mm diameter disk of EpiFix® modified placental tissue wasfound not to result in a significant migration of stem cells in vitro.However, 4 mm diameter EpiFix® modified placental tissue disks and 12×13mm square EpiFix® patches show a statistically significant increase inmigration of stem cells compared with control cells. One squarecentimeter of EpiFix® weighs 4 mg. Surprisingly, stem cell migrationeven in vitro requires a minimum mass of modified placental tissue toinduce migration, i.e. more than the mass of a 1.5 mm disk of EpiFix®modified placental tissue. Stated another way, the presence of asufficient amount of modified placental tissue correlates to asufficient concentration of stem cell recruiting factors such that stemcell recruitment is achieved. It is contemplated that higher amounts orconcentrations of stem cell recruiting factors (such as by using anextract comprising stem cell recruiting factors or addition of stem cellrecruiting factors to the modified placental tissue) will facilitatestem cell recruitment.

In addition, Example 4 describes in vivo implantation of a 5×5 mm squareEpiFix® modified placental tissue patch, leading to a statisticallysignificant increase in stem cell recruitment in mice, starting at about2 weeks post-implantation. In this regard, it is contemplated that theuse of a larger amount of EpiFix® modified placental tissue wouldfurther enhance stem cell recruitment either in a reduced time frame toachieve stem cell recruitment and/or increase the number of stem cellsrecruited over a given period of time. In various embodiments, theenhancement of stem cell recruitment is at least 5%, 10%, 20%, 30%, 40%,50%, 60%, 70%, 90%, 100% or more, when compared to the subject notreceiving a modified placental tissue graft. Regardless, in at leastthis example, the data shows that more than a minimal amount of EpiFix®modified placental tissue is required in order to effect stem cellrecruitment.

It will be appreciated that the actual amounts of placental tissue orplacental tissue extract administered in a specified case will varyaccording to the specific cardiac tissue to be treated, the particularcompositions formulated, the mode of application, and the degree ofdisease or injury in particular subject being treated. Dosages for agiven host can be determined using conventional considerations, e.g. bycustomary comparison of the differential activities of the subjectcompounds and of a known agent, e.g., by means of an appropriateconventional pharmacological protocol. Physicians and formulators,skilled in the art of determining doses of pharmaceutical compounds,will have no problems determining dose according to standardrecommendations (Physician's Desk Reference, Barnhart Publishing(1999)).

In some aspects, one or more stem cell recruiting factors that enhancestem cell chemotaxis and/or recruitment may be added to the modifiedplacental tissue of the present technology. In other aspects, stem cellrecruiting factors can be added to micronized placental tissue.Alternatively, stem cell recruiting factors may be added to layers of alaminate tissue graft. Thus, for example, cytokines, chemokines, growthfactors, extracellular matrix components and other bioactive materialscan be added to the modified placental tissue to enhance native stemcell recruitment. U.S. patent application Ser. No. 13/745,642, filed onJan. 18, 2013, titled “ISOLATED PLACENTAL STEM CELL RECRUITING FACTORS”,the content of which is incorporated by reference in its entirety,describes isolated stem cell recruiting factors, compositions comprisingthe isolated stem cell recruiting factors and methods for isolating thestem cell recruiting factors.

Specific non-limiting examples of stem cell recruiting factors mayinclude one or more of the following: CC chemokines, CXC chemokines, Cchemokines, or CX3C chemokines. Other stem cell recruiting factors mayfurther include growth factors such as cc-fibroblast growth factor (αFGFor αFGF-1), β-fibroblast growth factor (βFGF-1 or (βFGF-2),platelet-derived growth factor (PDGF), vascular endothelial growthfactor (VEGF-A, B, C, D or E), angiopoietin-1 and -2, insulin-likegrowth factor (IGF-1), bone morphogenic protein (BMP-2 and -7),transforming growth factor-α and -β (TGF-α and TGF-β), epidermal growthfactor (EGF), connective tissue growth factor (CTGF), hepatocyte growthfactor (HGF), human growth hormone (HGH), keratinocyte growth factor(KGF), tumor necrosis factor-α (TNF-α), leukemia inhibitory factor(LIF), nerve growth factor (NGF), stromal cell derived factor 1(SDF-1α), granulocyte macrophage colony stimulating factor (GM-CSF) andother factors as is known in the art.

In another aspect, the modified placental tissue is used in conjunctionwith conventional treatments, including, but not limited to, coronarybypass procedures, and may also be used in combination with matrices orscaffolds comprised of biocompatible materials, such as collagen,hyaluronic acid, gelatin or combinations thereof.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of howcompositions, and methods described and claimed herein are made andevaluated, and are intended to be purely exemplary and are not intendedto limit the scope of what the inventors regard as their invention.Efforts have been made to ensure accuracy with respect to numbers (e.g.,amounts, temperature, etc.) but some errors and deviations should beaccounted for. Unless indicated otherwise, parts are parts by weight,temperature is in ° C. or is at ambient temperature, and pressure is ator near atmospheric. There are numerous variations and combinations ofreaction conditions, e.g., component concentrations, desired solvents,solvent mixtures, temperatures, pressures and other reaction ranges andconditions that can be used to optimize the product purity and yieldobtained from the described process. Only reasonable and routineexperimentation will be required to optimize such process conditions.

Example 1—Preparation of Micronized Placental Tissue

Amnion/chorion tissue grafts used here to produce the micronizedparticles were produced by the process described in US 2008/0046095,which is incorporated by reference in its entirety. Tissue grafts (4cm×3 cm) and two 9.5 mm steel grinding balls were placed in 50 mL vialsand the vials subsequently sealed. The vials were placed in theCryo-block, and the Cryo-block was placed in a Cryo-rack. The Cryo-rackwas placed into a liquid nitrogen holding-Dewar flask. Tissue sampleswere subjected to vapor phase cooling for no more than 30-60 minutes.The Cryo-rack was removed from the Dewar flask, and the Cryo-block wasremoved from the Cryo-rack. The Cryo-block was placed into the Grinder(SPEX Sample Prep GenoGrinder 2010) and set at 1,500 rpm for 20 minutes.After 20 minutes had elapsed, the tissue was inspected to ensuremicronization. If necessary, the tissue was placed back into the Dewarflask for an additional 30-60 minutes, and moved to the grinder for anadditional 20 minutes to ensure sufficient micronization. Once thetissue was sufficiently micronized it was sorted using a series ofAmerican Standard ASTM sieves. The sieves were placed in the followingorder: 355 μm, 300 μm, 250 μm, 150 μm, and 125 μm. The micronizedmaterial was transferred from the 50 mL vials to the 355 μm sieve. Eachsieve 20 was agitated individually in order to thoroughly separate themicronized particles. Once the micronized particles were effectivelyseparated using the sieves, the micronized particles having particlesizes of 355 μm, 300 μm, 250 μm, 150 μm, and 125 μm were collected inseparate vials.

Example 2—Preparation of Tissue Grafts with Micronized Placental Tissue

Various modifications and variations can be made to the modifiedplacental tissue, compositions and methods described herein. Otheraspects of the modified placental tissue, compositions and methodsdescribed herein will be apparent from consideration of thespecification and practice of the modified placental tissue,compositions and methods disclosed herein. It is intended that thespecification and examples be considered as exemplary.

A detailed description of suitable cross-linking agents and proceduresis provided in concurrently filed U.S. Patent Application Ser. No.61/683,697, filed on Aug. 15, 2012 and entitled PLACENTAL TISSUE GRAFTSMODIFIED WITH A CROSS-LINKING AGENT AND METHODS OF MAKING AND USING THESAME which application is incorporated herein by reference in itsentirety.

A detailed description of reinforced placental tissue grafts is providedin concurrently filed U.S. Patent Application Ser. No. 61/683,699 filedon Aug. 15, 2012 and entitled REINFORCED PLACENTAL TISSUE GRAFTS ANDMETHODS OF MAKING AND USING THE SAME which application is incorporatedherein by reference in its entirety.

A detailed description of making and using micronized placental tissueand extracts thereof is provided in concurrently filed U.S. PatentApplication Ser. No. 61/683,700 filed on Aug. 15, 2012 and entitledMICRONIZED PLACENTAL TISSUE COMPOSITIONS AND METHODS OF MAKING AND USINGTHE SAME which application is incorporated herein by reference in itsentirety.

Example 3—Cell Migration in the Presence of EpiFix®

Human mesenchymal stem cells (human MSC) were evaluated in cell culturein the presence of samples of EpiFix® to determine whether the EpiFix®would induce migration of the human MSC. EpiFix® is a layer of amnionand chorion with the epithelial layer intact.

Materials and Methods

Standard migration assays were performed in 24-well cell culture insertswith 8-μm pore membrane filters at the bottom of the insert (see FIG. 1;BD Biosciences). 24 hours prior to the start of the experiment, humanMSCs (one donor, passage 3) were cultured in serum free media, and 300μL of 5 μg/mL fibronectin in PBS was placed into each cell cultureinsert to enable adsorption of fibronectin to the cell culture insertsurface overnight.

On the day of the experiment, 700 μL of serum-free culture medium wasloaded into the bottom wells of the plate, followed by the addition ofdifferently sized portions of sterilized EpiFix® (Low: 1.5-mm diameterdisk; Medium: 4-mm diameter disk; High: 12×13 mm square, trimmed into3-4 mm square pieces; n=6 EpiFix® tissue donors tested) (FIG. 2). Onesquare centimeter of EpiFix® weighs 4 mg. Serum-free medium and mediumwith 10% fetal bovine serum (n=6) acted as negative and positivecontrols, respectively. Human MSCs (40,000 cells in 300 μL) were thenloaded into the cell culture inserts and cultured for 24 hours. Then,both sides of the cell culture inserts were rinsed with PBS, andnon-migrating cells in the upper portion insert were removed with acotton-tipped applicator. Cells on the lower side of the insert plus themembrane filter were fixed in 10% formalin for 20 minutes, then rinsedand stained with hematoxylin for 5 min. The number of cells migratingthrough the membrane were counted on the lower surface of the membranewith an inverted microscope (Nikon TE2000; SPOT Software 4.6).

Data were normalized to the 10% FBS positive control and are expressedas mean±standard deviation of counted, migrated cells per 100× fieldmicrograph for each sample well. Statistical comparisons were performedusing a Box-Cox transformation to normalize data variance, followed byone-factor analysis of variance (ANOVA) with Tukey's honestlysignificant difference post-hoc test.

Results

The Low group (1.5 mm diameter disk) containing the smallest EpiFix®sample was not significantly different from the no serum negativecontrol (see bar graph in FIG. 2). Both the Medium group (4 mm diameterdisk) and the High group (12×13 mm square, trimmed into 3-4 mm squarepieces) were statistically higher than the no serum control (about 60%and 75% migration relative to control; see FIG. 2), indicating thatEpiFix® stimulated cell migration. The High group was not significantlydifferent from the Medium group. The results indicate that the EpiFix®product contains one or more factors that attract human mesenchymal stemcells.

Example 4—Stem Cell Recruitment in Mice Receiving EpiFix® Implants

A study was undertaken to determine whether EpiFix® implanted in normalmice caused recruitment of stem/progenitor cells, focusing on mousehematopoietic stem cells (HSCs) and mouse mesenchymal stem cells (mouseMSCs).

Materials and Methods

EpiFix® products from six donors were used for implantation in normalmice. A 5×5 mm square of EpiFix® was surgically placed subcutaneously in4 month old FVB/NJ mice (weighing between about 23.50 g and about 30 g).Four mice were implanted per sample per time point. The time points were3, 7, 14 and 28 days. The negative controls were normal skin and shamoperated mice (surgical incision but no implant). Decellularized dermalmatrix (acellular dermal matrix; ADM) was used as the comparativeimplant (Type I collagen, no cytokines). The implant and overlying skinwas harvested for fluorescence-activated cell sorting (FACS).

Implants and overlying skin were harvested, cut into 1 mm² sections, andincubated in a 0.15% dispase/0.075% collagenase solution at 37° C. for 1hour. After centrifugation, samples were stained with a lineage antibodycocktail as described below. CD31 antibody was added followed by AlexaFluor 647 anti-rat secondary antibody. Phycoerythrin-Cy7-conjugatedanti-CD45 antibody was incubated last. Samples were prepared andanalyzed as described below.

Samples were incubated with a lineage negative (lin−) antibody cocktail(Ter119/CD4/CD8a/Gr-1/CD45R/CD11b) followed by phycoerythrin-Cy5anti-rat secondary antibody. For mesenchymal stem cell analysis,conjugated antibodies were added against CD45 (phycoerythrin-Cy7) andSca-1 (fluorescein isothiocyanate). For hematopoietic stem cellanalysis, conjugated antibodies were added against CD45(phycoerythrin-Cy7), c-Kit (phycoerythrin), and Sca-1 (fluoresceinisothiocyanate). Samples were incubated with antibodies for 30 minutesand then washed by adding 5 volumes of 2% fetal bovine serum inphosphate-buffered saline with 2 mM ethylenediaminetetraacetic acid.Cells were centrifuged and then re-suspended in propidium iodide for 1minute at 4° C. Samples were analyzed using an LSR Flow Cytometer. UsingCellQuest software), samples were gated for lin⁻/Sca-1⁺/CD45⁺ to definemesenchymal stem cells and for lin⁻/Sca-1 Vc-Kie/CD45⁻ to definehematopoietic stem cells.

Results

Mouse HSCs were significantly increased following EpiFix® implantationcompared to negative controls at days 7, 14 and 28 (see FIG. 3A). MouseHSCs remained significantly increased in the EpiFix® samples at day 28compared to ADM.

Mouse MSCs were significantly increased following EpiFix® implantationcompared to negative controls at day 7 (see FIG. 3B). The averagepercentages of mouse MSCs were increased at all time points compared tonegative controls.

Thus the data described above show that EpiFix® implants effectivelyrecruit both HSCs and MSCs in vivo in normal mice. The data also showthat EpiFix® leads to longer term HSC recruitment than acellular dermalmatrix (ADM), supporting the hypothesis of a cytokine mediated effect ofEpiFix®.

Example 5—Stem Cell Characterization in Mice Receiving EpiFix® Implants

A study was undertaken to characterize stem cells recruited to EpiFix®implantation sites in mice, using flow cytometry andimmunohistochemistry.

Materials and Methods

Sterile, Purion® processed EpiFix® (obtained from MiMedx) in a 5×5 mmsquare patch was implanted subcutaneously through a skin incision on thebacks of sixteen 4 month old FVB/NJ mice. Identical skin incisions weremade in another sixteen mice to function as a control treatment (sham).For comparison with a collagen scaffold, a 5×5 mm square patch ofdecellularized human dermis (acellular dermal matrix; ADM) was implantedsubcutaneously on the backs of sixteen mice. Un-operated mice were usedas a source of “normal” back skin for the analyses.

The surgical site was removed at 3, 7, 14 and 28 days followingimplantation for analyses of stem cells. Four animals/group were used ateach time point. Stem cells were identified with two distinct methods:Fluorescence-activated cell sorting (FACS) and immunohistochemistry(IHC). For the FACS analysis, all cells were isolated from the amnionand associated regenerated tissue. The cells were fluorescently labeledwith antibodies to specific stem cell markers. The identity and numberof each cell type were determined with a flow cytometer.

For the immunohistochemical analyses, the membrane and associatedregenerated tissue was fixed, sectioned for slides, and stained withspecific antibodies to stem cells. Two antibodies were used for theimmunohistochemistry: anti-CD34, which specifically detectshematopoietic progenitor cells (HPC), and reacts with dermal progenitorcells, endothelial cells, dendritic cells; and anti-CD31, which detectsendothelial cells. The stained tissue sections were examinedmicroscopically and the presence and number of specific stem cell typeswere measured. For the experimental analysis, the relative number ofeach cell type was counted. The results were calculated as thepercentage of each cell type (no. of immunostained cells/total number ofcells). Two areas were analyzed immunohistochemically for cellrecruitment: the tissue surrounding the implant and the implant itself.

Results

Hematopoietic progenitor cell (HPC) levels were significantly elevatedin tissue surrounding EpiFix® implants at days 14 and 28 compared tonegative controls. Hematopoietic progenitor cells were significantlyincreased in the tissue surrounding the EpiFix® implant at days 14 and28 compared to collagen scaffold ADM control.

Progenitor cells were recruited into the EpiFix® implant. Intra-implanthematopoietic progenitor cells peaked at day 14 in the EpiFix® implant,and remained elevated at day 28. Average intra-implant hematopoieticprogenitor cells were increased in the EpiFix® implant at days 14 and 28compared to control ADM. Progenitor cells were not recruited into theADM control implant.

Vascularization of the EpiFix® implant steadily increased from day 14 today 28. The amount of new vessel formation in the EpiFix® implant wassignificantly greater than that in the ADM control on day 28.

These data establish that EpiFix® contains one or more factors thatrecruit both hematopoietic stem cells and mesenchymal stem cells to thesite of injury. More of these stem cells were found in the EpiFix®membrane and associated regenerated tissue than in the sham. EpiFix® wassignificantly more effective than the control decellularized collagenscaffold in recruiting progenitor cells to colonize the implant site.There were more progenitor cells in the EpiFix® membrane than in thecontrol collagen scaffold.

EpiFix® also induced new blood vessel formation in the associatedregeneratedtissue and the EpiFix® membrane itself. Vascularization inthe EpiFix® membrane was significantly higher than in the collagenscaffold control.

Example 6—Effects of AmnioFix and EpiFix® on Cardiac Repair

A study was undertaken to study the effects of AmnioFix® and EpiFix®membranes on cardiac repair after acute myocardial infarction (MI),including infarct size and cardiac remodeling. Both AmnioFix® andEpiFix® (obtained from MiMedx) are derived from the amniotic membraneand contain structural collagens and extracellular matrix proteins, aswell as a number of growth factors and cytokines, including PDGF-AA,PDGF-BB, TGFα, TGFβ, bFGF, EGF, VEGF, IL-10, IL-4, P1GF, TIMP-1, TIMP-2,AND TIMP-4.

Materials and Methods

The experiment was performed using immune-compromised NOD/SCID mice inan effort to eliminate any potential engagement of the immune systems inresponse to the xenograft AmnioFix® or EpiFix® membrane. The wild-typemice was used for comparison.

The left descending coronary artery (LAD) of the NOD/SCID or wild-typemice was ligated to induce MI. The MI mice were either treated with theAmnioFix Membrane® EpiFix® membrane or saline as a control. Eight weekspost treatment, the mice were analyzed for infarct size, cardiacremodeling, cell apoptosis and proliferation as well as stem cellmobilization.

Results

Both the AmnioFix® and the EpiFix® membrane treated mice showeddecreased infarct area following coronary artery ligation compared tocontrol (saline) treated mice (FIGS. 4A and 4B). There was nostatistically significant difference between the EpiFix® andAmnioFix®-treated mice in infarct size. Several mice did not recoverfrom the coronary artery ligation but this was due to surgicalmortality, rather than the post-surgical membrane treatment.

To determine if the decreased infarct size was due to recruitment ofc-kit positive stem cells from the bone marrow or locally from theheart, sections of the cardiac tissue were stained for c-kit andanalyzed. There was a significant increase in c-kit positive stem cellsboth in the region directly in contact with the membrane, as well asregions of the heart peripheral to the membrane (FIGS. 5A-5C). Thisindicates that cardiac repair present outside of the region that haddirect contact with the membrane (either the AmnioFix® or EpiFix®membrane) may be mediated by stem cells. This may contribute to theobserved overall decrease in the fibrous area of the heart.

Furthermore, there was a significant increase in the level of the cellproliferation marker Ki-67 in the membrane-treated hearts compared tothe control group (FIGS. 6A and 206B). The Ki-67 expression extendedbeyond the region with direct contact with the membrane into the distalheart tissue. This further supports the paracrine effects of theamniotic membrane products in stimulating cell proliferation and tissuerepair.

In addition, there was an increase in the number of blood vessels foundin the membrane-treated hearts as indicated by CD31 staining compared tothe control treated mice (FIGS. 7A and 7B). This indicates that themembrane treatment increased angiogenesis, resulting in improved bloodsupply, promoting cardiac repair.

Conversely, there was a significant decrease in apoptosis in themembrane treated hearts compared to the saline treatment as measured byTUNEL staining (FIGS. 8A and 8B). These data indicate that AmnioFix® andEpiFix® inhibit cell apoptosis and promote cell survival, perhapsthrough improved blood supply and other paracrine mechanisms.

In summary, Example 6 demonstrates that the treatment with eitherEpiFix® or AmnioFix® membranes protected the mouse hearts followingcoronary artery ligation-induced MI. Each of these membranes reduced theinfarct area to similar extents. This decrease in infarct size was seenirrespective of the type of mice treated (NOD/SCID vs. wild-type mice).The pro-repair effects were associated with elevated levels of c-Kit+stem cells, increased cell proliferation and decreased cell apoptosis.In addition, there was an increase in the mean numbers of vessels in thetreated hearts. These results show that treatment with EpiFix® orAmnioFix® improve cardiac repair following MI through multiple paracrineeffects. There is no significant difference in the efficacy of theEpiFix® compared to the AmnioFix® in these experiments.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the compounds, compositions and methods described herein.

Various modifications and variations can be made to the compounds,compositions and methods described herein. Other aspects of thecompounds, compositions and methods described herein will be apparentfrom consideration of the specification and practice of the compounds,compositions and methods disclosed herein. It is intended that thespecification and examples be considered as exemplary.

What is claimed:
 1. A composition for treating damaged cardiac tissue,said composition comprising dehydrated, cleaned, and disinfectedplacental tissue configured for non-obstructive placement to an areaapproximate to a damaged cardiac tissue in an amount sufficient toreduce damage and induce healing; said placental tissue comprising adehydrated amnion and a dehydrated chorion, said dehydrated amnionhaving an epithelial cell layer and/or a fibroblast layer; and whereinsaid composition is in the form of a patch; is in an injectable form; oris micronized.
 2. The composition of claim 1, wherein the dehydrated,cleaned, and disinfected placental tissue retains an effective amount ofstem cell recruiting factors, growth factors, and/or angiogenesisinducing factors.
 3. The composition of claim 1, wherein the dehydrated,cleaned, and disinfected placental tissue comprises one or more ofPDGF-AA, PDGF-BB, TGFa, TGFB, bFGF, EGF, VEGF, IL-10, IL-4, P1GF,TIMP-1, TIMP-2, and TIMP-4.
 4. The composition of claim 1, which is apatch.
 5. The composition of claim 1, wherein the dehydrated, cleaned,and disinfected placental tissue is micronized.
 6. The composition ofclaim 1, which is in an injectable form.